FR2488908A1 - PROCESS FOR THE PREPARATION OF ACRYLAMIDE USING IMMOBILIZED CELLS OF A NEW TYPE - Google Patents

Abstract

PROCESS FOR PREPARING ACRYLAMIDE FROM ACRYLONITRILE IN AN AQUEOUS MEDIUM CONTAINING PRACTICALLY NO SALTS BY ACTION OF A MICRO-ORGANISM PRESENTING THE NITRILASIC ACTIVITY. THE MICRO-ORGANISM IS IMMOBILIZED IN A POLYMER CATIONIC ACRYLAMIDE-BASED GEL.

Description

The present invention relates to a process for the preparation of

  acrylamide using immobilized cells that are prepared by immobilizing microorganisms with activity

  nitrilase on a cationic polymer gel based on acrylamide.

  Acrylamide is a compound which has multiple applications for example as a starting material for the preparation of various polymers used themselves as flocculants, additives to base oils, polymers for recovery

  oil, and in other similar applications.

  Among the processes for the preparation of acrylamide, there has previously been described a process which comprises reacting acrylonitrile with water in the presence of a catalyst containing

  copper in the reduced state. However, this process has disadvantages

  various people; thus, for example, the preparation of the catalyst is complicated; difficulties are encountered in the regeneration of spent catalyst; and there are also complications in the separation and purification of the acrylamide formed. Furthermore, it is sought to obtain acrylamide under mild reaction conditions because the compounds containing double bonds in

  the molecule, like acrylamide, polymerize easily.

  It has already been considered highly desirable to develop a process for preparing acrylamide by hydrolyzing acrylonitrile using microorganisms in

conditions.

  It has long been known that micro-organisms

  nitrilase activity can be used to effectively hydrolyze

  acrylonitrile acrylamide. Among these microorganisms, those belonging to the genera Bacillus, Bacteridium in the sense of Prevot, are already known. Micrococcus and Brevibacterium in the sense of Bergey,

  etc. (see, for example, United States Patent No. 4,001,081).

  It has also been found previously that microorganisms

  Corynebacterium and Nocardia could be used for the hydrolysis of acrylonitrile (see, for example,

  United States of America No. 4,248,968).

  To prepare acrylamide from acrylonitrile using these microorganisms, acrylonitrile is contacted with the microorganisms or with immobilized cells of these microorganisms which have been prepared by immobilizing the cells. on polymeric gels, in an aqueous medium such as pure water, physiological saline, or a phosphate buffer solution. Recently, a continuous or discontinuous column process exploiting cells immobilized on granules has been widely exploited, in order to prevent the elution of impurities from the cells, to improve the separation of the cells and the solution. of reaction, to allow the repeated use of cells and to increase the

  enzyme stability. These methods exploit real cells

  granules have economic advantages. As a result, there has been proposed a process for the preparation of acrylamide by continuous reaction on a column in which

  immobilized microorganism cells that are prepared in

  cells in a polyacrylamide gel or substance

  analogous (see, for example, U.S. Patent No. 4,248,968).

  However, in the processes as proposed above, the use of a physiological saline solution, a phosphate buffer solution or a similar solution as an aqueous medium leads to the introduction of large amounts of sodium chloride, phosphates or similar substances in the aqueous acrylamide solution obtained which has drawbacks with regard to the quality of the desired product. Especially for

  the preparation of polymers based on high molecular weight acrylamide

  However, the presence of phosphates in acrylamide can lead to insolubility properties in the water of the polymers formed. So that to eliminate these salts, it is essential to carry out subsequent treatments such as ion exchange treatments. The expected advantages of the preparation of a high quality aqueous acrylamide solution are then lost without purification.

  special feature, which is a feature of the prepa-

  acrylamide by the immobilized cell technique.

  Thus, the advantages of the immobilized cell method are lost

  as an inexpensive process for the preparation of acrylamide.

  On the other hand, if a saline solution, a phosphate buffer solution or the like solution is not used as the aqueous medium, the immobilized cells swell during the hydration reaction and there is rapid loss. of the enzymatic activity of the cells. In addition, in the case of the column reagent, when an aqueous solution of acrylonitrile is passed over a column packed with immobilized cells. according to the conventional technique in polyacrylamide, the cells immobilized in the column swell rapidly after starting the hydration reaction and it becomes impossible to continue

effective operations.

  Although it is not fully understood why the immobilized cells swell during the hydration reaction, it is believed that this phenomenon is due to the repulsive forces produced between the negatively charged cells as the substrate solution passes and unlike osmotic pressure between the outside and the inside of the immobilized cells resulting from the difference between the concentrations of acrylonitrile and acrylamide between the outside and the inside of these cells, differences in concentration which

  produce when acrylonitrile enters the living cells

  and converted (hydrated) to acrylamide that migrates out of the immobilized cells. It is further believed that the degradation of enzymatic activity by the swelling phenomenon is due to the

  the enzyme is likely to escape from the living cells.

  because of swelling and that the stable conformation of normal cells in which the enzyme

is not inflated.

  As a result, it is believed that when the reaction is carried out in an isotonic medium such as saline solution, phosphate buffer solution, etc., no

  great difference in osmotic pressure between the outside and the inside

  immobilized cells, so that both the immobilized cells can be prevented from

the enzyme in a stable state.

  Following extensive research aimed at preparing

  immobilized cells that do not swell in the course of the hydration reaction, even when an aqueous solution of substrate containing no salt is used as aqueous medium, unlike the solutions described above, and which have excellent In view of the enzyme stability, we have found that such immobilized cells can be obtained by using a cationically rendered immobilized polymeric carrier to improve its affinity for cells or the enzyme. The invention accordingly relates to a process for the preparation of acrylamide from acrylonitrile using immobilized microorganism cells in which the immobilized cells are prepared by immobilizing a microorganism having the nitrilase activity on a cationic polymer gel based on acrylamide, and the acrylonitrile is brought into contact with the immobilized cells in an aqueous medium containing substantially no

no salt.

  Any microorganism capable of hydrolyzing acrylonitrile to acrylamide can be used in the invention regardless of their taxonomic classification. So, for example, we

  preferably uses strain N-771 belonging to the genus Corne-

  bacterium, as described in U.S. Patent No. 4,248,968 and filed at the Fermentation Research Institute, The Japanese Agency of Industrial Science and Technology under the number

  FERM 4445, the N-774 strain belonging to the genus Horn-

  bacterium deposited under the accession number FERM 4446 and the strain N-775 belonging to the genus Nocardia and filed under the accession number FERM 4447. It is also advantageously used in

  the invention the microorganisms described in the US Pat.

United States of America No. 4,001,081.

  For the preparation of immobilized cells with these microorganisms, they can be used in any of the following forms: culture medium containing the cells (culture broth), washed cells (residual cells),

  broken cells, and in analogous forms.

  The cationic polymer based on acrylamide as used in the invention for immobilization is a copolymer of acrylamide, a cationic monomer with ethylenic unsaturation copolymerizable with acrylamide, and a crosslinking monomer. soluble

in water.

  Examples of cationic monomers with ethylenic unsaturation copolymerizable with acrylamide include dialkylaminoalkyl acrylates, dialkylaminoalkyl methacrylates, dialkylaminoalkylacrylamides, dialkylaminoalkylmethacrylamides and their quaternary salts, and for example dimethyl methacrylate.

  thylaminoethyl, diethylaminoethyl methacrylate, dimethyl-

  aminopropylmethacrylamide, diethylaminopropylmethacrylamide and their quaternary salts formed with dimethyl sulfate, methyl chloride and the like. These monomers can

  be used alone or in combination with each other.

  Examples of water-soluble crosslinking monomers are methylenebisacrylamide, 1,3-di-acrylamidomethyl-2-imidazolidone, diacrylamidomethylethylene-urea, diacrylamidoethyl ether, ethylene glycol diacrylate, dimethacrylate and the like.

  Ethylene glycol and hexahydro-1,3,5-triacyl-S-triazines.

  With regard to the amounts of the three monomers used, the amount of acrylamide represents from 50 to 95%, the amount of ethylenically unsaturated cationic monomer from 1 to 50% and the amount of water-soluble crosslinking monomer represents

  from 0.1 to 20% of the total weight of the three monomers.

  The immobilized cells used in the process according to the invention are prepared, for example, by a process in which a suspension of the microorganism cells and the three monomers described above is mixed and polymerized in the presence of a catalyst. polymerization of the type commonly used in conventional processes such as potassium persulfate and dimethylaminopropionitrile, at pHs of 5 to 10, and preferably from 6 to 8, at a temperature of 0 to 30 C, and preferably from 0 to 30.degree. C, in a period of 30 to 60 min; a gel is thus obtained in which

  the cells are immobilized and imprisoned.

  The amount of the microorganism cells contained in the gel as obtained above is usually from 0.1 to 50%, and preferably from 1 to 20%, of the total weight of the aqueous gel; however, this amount may vary depending on the type of microorganism, its form of use and similar factors. The monomer content of the reaction solution ranges from 2 to 30%, and preferably from 5 to 20%,

the total weight of the solution.

  However, to prepare the gel containing the immobilized cells and in addition to the above process in which a cationic component in the monomer state is added and then the gel is formed by copolymerization, another process may be used in which all or a part of the cationic monomer is polymerized beforehand in the form of a water-soluble polymer which can still be gelled, and the water-soluble polymer is then mixed with the suspension of the cells of the microorganism containing the monomers; it is reacted to obtain the desired gel. It is also possible to prepare the water-soluble polymer to be added before the preparation of the gel by copolymerizing the cationic monomer with a portion of the acrylamide.

  Immobilized cells as described above are -

  put in the form of particles, if necessary after having been subjected to a treatment with a water-soluble dialdehyde such

than glutaric aldehyde.

  In the practice of the invention, the cells immobilized on a cationic support as described above can be crushed into particles to the desired size and introduced into a reactor

  or a column. By putting in contact with the immovable cells

  bilize an aqueous solution of acrylonitrile at a salt concentration of 0.1% or less by weight, and preferably 0.01 by weight or less, that is to say an aqueous solution of acrylonitrile containing substantially no salt, the desired acrylamide can be prepared. By correctly choosing the amount of immobilized cells used in the reaction, the concentration of acrylonitrile, the flow rate of the aqueous substrate solution and the like factors, a conversion of almost 100% can be achieved. In one

  such case, to maintain the nitrilase activity of the immo-

  During prolonged periods of time and to prevent the formation of secondary products such as acrylic acid, it is preferable to maintain an acrylonitrile concentration of 5% by weight or less, to observe a reaction temperature as low as possible in a an interval in which the aqueous solution of substrate does not freeze, that is to say to operate in a range from a point just above the freezing point to 100C

about, with a fold of 7.0 to 8.5.

  According to the invention, the reaction can be continued without swelling immobilized cells in the course

  of the reaction, although no physiological serum solution is used.

  A phosphate buffer or analogous solution as an aqueous solution of the substrate, and this, in addition, keeping the enzyme activity stable for extended periods. In particular, in the case of a continuous reaction on a column, the process according to the invention, in addition to the advantages described above, provides another advantage: it is possible to avoid any polymerization of the acrylamide formed (which would tend to occur in the course

  of the reaction) and the operations can be stabilized.

  It is therefore possible to obtain in the reaction effluent a transparent and colorless aqueous solution of acrylamide. Because this

  The solution contains practically no salt and almost no

  troublesome for the polymerization of acrylamide, it can be used as such or after concentration as a material

  first of the preparation of acrylamide polymers intended for

  even for use as flocculants, oil additives

  basic, and in similar applications.

  The examples which follow illustrate the invention without, however, limiting its scope; in these examples, the parts and percentages are by weight unless otherwise indicated. The concentrations of acrylonitrile, acrylamide and acrylic acid were determined by gas chromatography, EXAMPLE 1 and COMPARATIVE EXAMPLE 1 50 parts of washed cells (dry cell content 20%) of the cultured strain N-774 were mixed. aerobic culture medium (at pH 7.2) containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract with

  4.2 parts of acrylamide, 0.4 part of the quaternary salt of methacrylate

  dimethylaminoethyl and methyl chloride, 0.4 part of methylenebisacrylamide and 30 parts of 0.05 M phosphate buffer (pH 7.5, all the phosphate buffers described below are at the same pH); thus a homogeneous suspension is formed. At this suspension, we

  5 parts of a 5% aqueous solution of dimethylaminopropio-

  nitrile and 10 parts of a 2.5% aqueous solution of potassium persulfate and polymerized maintaining a temperature of 10 C or less for 1 hour; a gel containing the cells is obtained. This bulky gel containing the cells in the form of fine particles is put with the aid of a mixer (model VA-IOP of the firm Hitachi Ltd.) and mixed with 300 parts of 0.05 M phosphate buffer and 0 5 parts of a 50% aqueous solution of glutaraldehyde; treated with glutaraldehyde at 10 C or less for 30 min with stirring. The immobilized cells are washed with water and

  uses them for the preparation of acrylamide from acrylamide.

lonitrile as described below.

  Ten parts of the immobilized cell sample are mixed with 90 parts of water. To the mixture obtained, the acrylonitrile is intermittently added dropwise at a rate of 2 parts per hour while maintaining the pH at 7.5 with stirring; it is reacted with acrylamide formation at 10 C for 6 h. The reaction is almost quantitative and 110 parts of a 14.6% aqueous solution of acrylamide are obtained. After repeating the operation, determine

  the residual activity of the immobilized cells.

  By way of comparison, immobile cells are prepared

  The results are based on a polyacrylamide of the conventional type, that is, the comparative immobilized cells are prepared as described above, but with 4.6 parts of acrylamide and 0.4 part of methylenebisacrylamide. These immobilized cells are used for the same acrylamide formation reaction. When the reaction is complete, the residual activity of the

immobilized cells.

  The results obtained are reported in Table I

below.

  The residual activity of the immobilized cells was determined as follows: The sample of immobilized cells is ground entirely in the mortar and then diluted with 0.1 M phosphate buffer, to form a suspension with a cell content. 0.1%. To 5 ml of the suspension is added 5 ml of a 5% aqueous solution.

TABLE I

  Residual activity,% Number of repetitions of the reaction

Example 1

  Comparative Example 10 to 5 N Co Co% 0 t Co

TABLE II

Example

3 4 5 6 7 8

  Comparative Example Composition of the polymer for immobilization of the cells. % Acrylamide

Dimethyl methacrylate

  aminoethyl Methylenebis-acrylamide Residual activity, Y. Number of repeats of the reaction: fi Il * l

94 90 85 80 70 50

1 5 10 15 25 45

5 5 5 5 5

100 100 100 100 100

95 95 95 95 80

85 85 85 80 65

80 80 80 76 60

o F ru o o Co N0 O>

TABLE III

Example

  Comparative 9 10 11 13 13 Composition of the polymer for immobilization

cells, 7.

  Acrylamide Quaternary salt of dimethylethyl methacrylate and methyl chloride Dimethylaminopropylmethacrylamide Methylenebisacrylamide 1,3-Di-acrylamidomethyl-2-imidazolidone Polymer of the quaternary salt of methyl chloride and dimethylaminoethyl methacrylate (MW about 500,000)

  Polymer of dimethyl methacrylate

  aminoethyl (Mw about 1,000,000) Copolymer of acrylamide and quaternary salt of methyl chloride and dimethylaminoethyl methacrylate (MW about 500,000) Copolymer of acrylamide and dimethylaminoethyl methacrylate (MW about 2,000,000) Residual activity ,% Number of repetitions of the reaction O "i 1 It 3

"I 5

82 75 75 75 75

O 10 - -

- 5 5 5

R - - -

- - - 20

- - - - 20

Example

j- pu Co Co c, w

Claims (10)

R E V E N D I C A T I 0 N S CLAIMS   A process for the preparation of acrylamide from acrylonitrile in an aqueous medium containing substantially no salts by the action of a microorganism having a nitrilase activity, characterized in that the microorganism is immobilized   in a cationic polymer gel based on acrylamide.   2. Process according to claim 1, characterized in that   that the cells of microorganisms are immobilized by polymeric   of a mixture of acrylamide, a cationic monomer with unsaturated   ethylenic ration copolymerizable with acrylamide, and a water-soluble crosslinking monomer in an aqueous suspension of microorganisms   possessing the nitrilase activity.   3. Method according to claim 1, characterized in that   that the cells of microorganisms are immobilized by polymerisation   mixture of acrylamide and a hydrophilic crosslinking monomer.   or a mixture of acrylamide, an ethylenically unsaturated cationic monomer copolymerizable with acrylamide and a water-soluble crosslinking monomer in an aqueous suspension of the microorganism having the nitrilase activity in the presence of a cationic water-soluble polymer.   4. Method according to claim 2 or 3, characterized in that the ethylenically unsaturated cationic monomer copolymerizable with acrylamide consists of at least one monomer chosen from   group formed by dimethylaminoethyl methacrylate, methacrylate   diethylaminoethyl late, dimethylaminopropylmethacrylamide,   diethylaminopropylmethacrylamide and their quaternary salts.   5. Process according to claim 3, characterized in that the water-soluble cationic polymer consists of at least one   selected from the group consisting of homopolymers of metha-   dimethylaminoethyl crylate, diethylaminoethyl methacrylate,   dimethylaminopropylmethacrylamide, diethylaminopropylmethacryl-   amide and their quaternary salts and copolymers of one or   several of these monomers and acrylamide.   6. Method according to claim 2 or 3, characterized in that the polymer gel containing the immobilized microorganisms contains 50 to 95% by weight of acrylamide, 1 to 50% by weight of the cationic monomer and 0.1 to 20% by weight of the crosslinking monomer relative to the total weight of the three monomers.   7. Process according to any one of claims 1 to 3,   characterized in that the microorganism content of the gel containing   the immobilized microorganisms is from 0.1 to 50% by weight.   8. Process according to claim 7, characterized in that the microorganism content of the gel containing the microorganisms   immobilized is from 1 to 20% by weight.   9. Process according to claim 2 or 3, characterized   this is carried out at a pH of 5 to 10 and at a temperature of 0 to 30 C.   Method according to claim 2 or 3, characterized in   this is carried out at a pH of 6 to 8 and a temperature of 0 to 15 C.